This invention relates to a method, based on an immunological approach, for detecting the presence of a Mycobacterium species and a kit and antibodies for use therein.
The various known species of Mycobacterium are known to cause a number of infectious diseases in humans and animals, One such Mycobacterium species, Mycobacterium tuberculosis, causes tuberculosis in humans.
Tuberculosis is considered to be the major communicable disease throughout most of the world. Despite great advances in medical science and a range of effective drugs, which for some time created the impression that the disease had been conquered, and despite organized international efforts, tuberculosis remains a world health problem of staggering proportions. More than 8 million new cases world-wide and more than 3 million deaths were reported in the year 1990 alone (Snider, 1994). Predictions made by the World Health Organisation indicate that by the year 2000 the annual figures will grow to 10.2 million new cases and 3.5 million deaths, with Asia and Sub-Saharan Africa being the most affected continents (Dolin, Raviglione and Koch, 1994). The global distribution of the estimated number of tuberculosis cases for the current decade and the estimated number of deaths for the same period, is presented in Dolin, Raviglione and Koch, 1994.
The close association documented between tuberculosis and AIDS as well as the frequently concomitant presence of both these diseases add gravity to the situation (Torres et al., 1990; De Cock, 1994; Cantwell and Binkin, 1994; Murray, 1994). The emergence of multiple-drug resistance among the strains of Mycobacterium tuberculosis and other atypical mycobacteria has introduced an additional dimension to this gigantic problem (Blumberg, Miller and Koornhof, 1994; Morse, 1994).
The accurate and timely detection of tuberculosis and related mycobacterial diseases is one of the important requirements to develop a more successful global strategy to combat these diseases.
Traditional laboratory detection methods have major disadvantages of either not being capable of distinguishing between live and dead bacilli (the quick and simple Ziehl-Neelsen staining) or, if these methods confirm the presence of the live bacilli (direct cultivation), a number of weeks is required before the laboratory tests are completed. This, in turn, may delay the commencement of treatment and may lead to further spread of the disease.
The more recent approaches are based either on the detection of patients' response to the infection by methods such as serological tests, lymphocyte proliferative responses to mycobacterial antigens or by determining the level of adenosine deaminase, or on the detection of mycobacterial antigens and constituents using immunoassays such as ELISAs, gas and liquid chromatography or mass spectrophotometry. More modern, molecular approaches include polymerase chain reaction (PCR), DNA probes or DNA fingerprinting (Musial et al., 1988; Godfrey-Faussett, 1994).
Mason et al, 1993 (Tubercle and Lung Disease) describe a method of identification and characterization of mycobacterial species by using monoclonal antibodies inter alia directed to mycobacterial lipoarabinomannan and unspecified glycolipids.
Wiker et al, 1991 (Journal of General Microbiology) describe a method for characterizing and identifying mycobacterial species grown in culture by detecting characteristic protein antigens of the mycobacteria.
Hamid et al, 1993 (Journal of General Biology) describe a text to distinguish Mycobacteria from other bacteria by precipitating mycolic acids and performing a TLC analysis on them.
Young, 1980 (Journal of General Biology) describe a method for extracting and analyzing mycolic acids from mycobacteria by TLC and shows a difference in the mycolic acid profile between Mycobacterium leprae and other bacteria in leprosy biopsies.
European Patent Application No 0407605 details a method of detecting antibodies in patient sera to bacteria from the species Nocardia by detecting antibodies raised to nocardial mycolic acids.
However, none of the methods listed above fulfills all the requirements for a quick, simple and reliable test capable of distinguishing between live and dead mycobacteria cells.